Project Summary Management of ongoing pain not only improves patient comfort but also accelerates postoperative recovery and diminishes the likelihood of developing chronic pain, ultimately reducing the cost of care. Many types of pain at rest (spontaneous or ongoing pain) are likely driven by ongoing activity (OA) in nociceptors that occurs in the absence of a discrete stimulus. In a model of neuropathic pain, nociceptive OA is produced by a prolonged depolarization of resting membrane potential, reduction of the action potential threshold, and an increased incidence of large depolarizing spontaneous fluctuations (DSFs) of the membrane potential. A low dose of the inflammatory mediator serotonin (5-HT), when combined with artificial depolarization, strongly potentiates the generation of large DSFs and OA in probable nociceptors from uninjured rats, showing that DSFs can also be enhanced acutely to promote OA. A variety of 5-HT receptors are expressed in sensory neurons, and it is unknown which of these receptor types and downstream pathways are important for 5-HT potentiation of DSFs. Previous studies demonstrate that AKAP-scaffolded PKA activity is important for maintenance of OA in nociceptors after spinal cord injury. The T-type voltage-gated Ca2+ channel Cav3.2 is stimulated by PKA and by Gs-coupled 5HT7 receptor activity, and PKA-dependent internalization of Slack KNa channels induces nociceptor hyperexcitability. These and other observations led to the hypothesis that peripheral 5-HT modulates specific Ca2+ (T-type), and K+ (KNa) conductances via PKA to generate large DSFs that promote OA in nociceptors and ultimately ongoing pain. This hypothesis will be tested in three specific aims. In Aim 1, we will determine the receptors and cell signaling mechanisms mediating 5-HT potentiation of DSFs and OA. Using available pharmacological tools along with patch clamp and high content microscopy techniques, we will test the hypothesis that 5-HT enhancement of DSFs is mediated largely by PKA activation downstream of Gs-coupled 5- HT receptors. In Aim 2, the specific conductances important for 5-HT-dependent generation of large DSFs and OA will be defined. Using patch clamp recording and pharmacology, we will probe the necessity and sufficiency of increased T-type Ca2+ and TRPC channel conductances and inhibition of KNa channel conductance for generation of large DSFs. Our mechanistic model for enhancement of the generation of large DSFs and OA may be particularly relevant to deep tissue incision pain in which platelet aggregates release 5-HT at and near a wound. Deep tissue incision has been shown to induce both OA in nociceptors and spontaneous pain behavior for a few days after injury. It is unknown whether 5-HT or PKA at or near the incision site plays a role in the OA and ongoing pain following incision. In Aim 3, we will determine whether peripheral 5-HT and PKA contribute to pain behavior in a model of postoperative pain. This proposed study will contribute new insight into the molecular mechanisms underlying pain-related ongoing activity in nociceptors and the contributions of peripheral serotonin and PKA to ongoing and evoked pain in a postoperative pain model.